Project Summary Metastasis, the dissemination of cancer cells from the primary tumor to secondary sites, is the leading cause of cancer-related mortality. Although chemotherapy improves overall survival in early breast cancer, it does not provide definite cure for metastasis. Paradoxically, chemotherapy induces pro-metastatic changes in the tumor microenvironment if given in the pre-operative (neo-adjuvant) setting. These pro-metastatic changes are primarily initiated via infiltration of tumors by pro-angiogenic Tie2Hi macrophages, which are essential components of cancer cell intravasation sites called Tumor MicroEnvironment of Metastasis (TMEM). Only invasive and migratory cancer cell expressing MenaINV can disseminate via TMEM. In mouse mammary carcinoma and patient derived xenografts, neo-adjuvant chemotherapy increases (i) TMEM assembly, (ii) the density of cancer cells expressing MenaINV, (iii) the number of circulating tumor cells, and (iv) lung metastases. This proposal will elucidate molecular mechanisms by which chemotherapy exerts these pro-metastatic changes in the breast tumor microenviroment, so that predictive biomarkers and targetable signaling pathways of ?chemotherapy-induced metastasis? can be identified. We hypothesize that macrophages recruited to tumors upon neo-adjuvant chemotherapy educate tumor cells not only to become dissemination-competent by inducing MenaINV expression through juxtacrine interactions, but also to obtain tumor-initiating capabilities responsible for tumor growth initiation at distant sites. In Aim 1, we will investigate whether macrophage-cancer cell interaction is required for induction of MenaINV and MenaINV-mediated pro-metastatic phenotypes upon treatment with neo-adjuvant chemotherapy. This will be accomplished using established macrophage depletion studies in vivo, coupled with advanced microscopy. In Aim 2, we will investigate whether breast tumor cells expressing MenaINV-Hi also harbor stem cell capabilities. MDA-MB-231 cells expressing a fluorescent stem cell reporter (SORE6) will be used to generate xenografts for multiphoton intravital imaging microscopy and fixed- tissue multichannel immune-fluorescent microscopy and study the effects of neo-adjuvant chemotherapy on tumor cell stemness. Aim 3 is designed to unravel the signaling pathways responsible for the aforementioned pro-metastatic phenotypes. Transgenic and pharmacological inhibition mouse models targeting the relevant juxtacrine pathways will be developed to eliminate macrophage-tumor cell interactions and study whether these pathways are instrumental in chemotherapy-induced metastasis. The research environment at the Albert Einstein College of Medicine offers outstanding opportunities for collaborations, scientific discussion and career development. The proposed studies and career development training coupled with an exceptional team of Dr. Condeelis, expert in advanced microscopy, Dr. Oktay, physician scientist, Dr. Wakefield a pioneer in stem cell research, and Dr. Richard Stanley, leader in macrophage biology, will ensure that I achieve my goal of establishing a successful, independently-funded lab at a major university.